The invention relates to a scanning microscope with a first and at least one other detection channel, whereby the first detection channel comprises at least one first detector and the other detection channel at least one other detector to detect a detection light beam given off by a sample.
The invention additionally relates to a method for examining a sample using a scanning microscope.
In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflection or fluorescent light emitted by the sample. The focus of an illumination light beam is moved in an object plane with the help of a controllable beam deflector, generally by tipping two mirrors, whereby the axes of deflection are usually positioned perpendicular to each other, so that one mirror deflects in the x-direction and the other in the y-direction. The mirrors are tipped with the help, for example, of galvanometric positioners. The power of the light coming from the object is measured dependent on the position of the scanning beam. Generally, the positioners are provided with sensors to determine the actual position of the mirrors.
In confocal scanning microscopy in particular, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optic with which the light from the source is focused on a pinhole aperture—the so-called excitation aperture—, a beam splitter, a beam deflector to control the beam, a microscope optic, a detection aperture, and detectors to detect the detection or fluorescent light. The illumination light is often coupled via the beam splitter which, for example, may be implemented as a neutral beam splitter or as a dichroic beam splitter. Neutral beam splitters have the disadvantage that a great deal of excitation or detection light is lost, depending upon the splitting ratio.
The fluorescent or reflection light coming from the object goes back to the beam splitter via the beam deflector, passes through it, and finally focuses on the detection aperture, behind which are the detectors. Detection light that does not originate directly from the focal region takes another light path and does not pass through the detection aperture, so that pixel information is obtained that leads to a three-dimensional image as a result of sequential scanning of the object. In most cases, a three-dimensional image is achieved by layered data imaging, whereby the path of the scanning light beam ideally describes a meander on or in the object. (Scanning a line in the x-direction at a constant y-position, then interrupting x-scanning and y-repositioning to the next line to be scanned, and then scanning this line at a constant y-position in negative x-direction, etc.). To enable layered data imaging, the sample table or the objective is repositioned after scanning a layer so that the next layer to be scanned is brought into the focal plane of the objective.
In many uses, samples are prepared with several markers, such as several different fluorescent dyes. These dyes can be sequentially excited, for example by illumination light beams exhibiting different excitation wavelengths. Simultaneous excitation by an illumination light beam that comprises light of several excitation wavelengths is also the norm. An arrangement with a single laser that emits several laser lines is known from the European patent application
EP 0 495 930, “Confocal Microscope System for Multiple Color Fluorescence.” Currently, such lasers are mostly implemented as mixed gas lasers, particularly as ArKr lasers.
Multiple band detectors are often used to detect detection light coming from the sample. A device to select and detect at least two spectral regions of a light beam with a selector and detector is known from published application DE 433-0347 A1. The device is designed for reliable and simultaneous selection and detection of varying spectral regions at high yield and with the simplest construction such that the selector component for spectral fanning out of the light beam—for example a prism or a grid—and a means for blocking a first spectral region, on the one hand, and for reflecting at least a portion of the non-blocked spectral region, and the detector comprises a first detector arranged in the beam path of the blocked spectral region and a second detector arranged in the beam path of the reflected spectral region. Preferably, a slit diaphragm with mirrored aperture walls is implemented to block out a first spectral region and on the other hand to reflect at least a portion of the non-blocked spectral region. In particular, the device may be used as a multiband detector in a scanning microscope.